Transcription control element for increasing gene expression in myoblasts

ABSTRACT

A transcription control element is provided for controlling gene expression in myogenic cells. The transcription control element comprises an isolated DNA segment having an enhancer activity in cultured cells and in non-cultured myogenic cells. The transcription control element is isolated from upstream regions of genes encoding bHLH myogenic regulatory proteins. Specifically, an enhancer element from the upstream region of human myoD and an enhancer element from the upstream region of a quail qmf1 are provided. These myoblast-specific transcription control elements are capable of significantly increasing the levels of gene expression in myogenic cells and are intended to be applied in gene therapy, using myoblast transfer and microinjection techniques, wherein myoblast-specific gene expression is desired or required.

Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health.

This application is a 371 of PCT/US93/02767, filed Mar. 24, 1993, whichis a continuation of 07/866,386, filed Apr. 10, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of gene expression and genetherapy. Specifically, a transcription control element is provided forcontrolling gene expression in myogenic cells.

BACKGROUND

Vertebrate skeletal muscle fibers are formed by the cellular fusion ofprogenitor myoblasts, which are embryonic cells that proliferate andpopulate the muscle-forming regions of embryos. Myogenic lineages becomedetermined during somite morphogenesis, leading to the formation ofstably determined myoblasts.

The process of myoblast differentiation into muscle fibers has beeninvestigated in cell cultures of clonal embryonic myoblasts andestablished myoblast cell lines. In dispersed cell cultures, myoblastscan proliferate clonally in the presence of medium rich in growthfactors, but retain their potential to differentiate in fused musclefibers. Thus, myoblasts are a stably determined cell type, capable ofextensive cell division, the progeny of which faithfully inherit theirmyoblast identity and can express their potential to differentiate intomuscle fibers. The growth and differentiation of myoblasts is controlledby extracellular factors, specifically growth factors such as basicfibroblast growth factor (bFGF) and transforming growth factor-β(TGF-β). In the presence of such growth factors, myoblasts proliferate,whereas in reduced concentrations of such factors, myoblasts can existin the cell cycle in G₁, fuse and differentiate into contractile fibers.

Myogenesis, therefore, involves "determination" of the myoblast lineagesin the somite and "differentiation" of myofibers in the muscle-formingregions of the embryo. The molecular mechanisms regulating cell lineagedetermination have been studied using the mouse cell line C3H1OT1/2(10T1/2). Konieczny and Emerson, Cell, 38: 791 (1984). This model cellculture system has allowed identification of several mammalian genes(myoD, myogenin, Myf-5 and MRF-4) that regulate the determination of theskeletal lineage. These genes encode transcription factors comprising asubgroup within the basic helix-loop-helix (bHLH) superfamily ofMyc-related DNA binding proteins.

It has been determined that the bHLH myogenic regulatory proteins areevolutionarily conserved, as evidenced by amino sequence homology.Pownall et al., Seminars in Devel. Biol., 3: 229-241 (1992); de laBrousse et al., Genes and Devel. 4: 567-581 (1990). Specifically, it hasbeen shown that the protein qmf1, a myoD protein from quail (QMyoD)shares extensive homology with mouse MyoD1, Myf5, myogenin and a MyoD1sequence from Xenopus Laevis (XMyoD). de la Brousse et al., supra.

As transfected cDNAs, the aforementioned myogenic regulatory factorsinduce myogenic conversion of multipotential 10T1/2 cells to stablydetermined populations of proliferative myogenic cells. Consistent withtheir function in determination, these myogenic regulatory genes areexpressed exclusively in skeletal muscle lineages of the embryo,beginning at the early stages of somite formation. Although thesetranscription factors regulate the determination of skeletal musclelineage, they themselves are also regulated. The transcriptionalregulatory mechanisms that activate their expression in the skeletalmuscle lineage of the embryo have heretofore remained unknown.

Because myoblasts are proliferative (i.e., regenerative) and are capableof fusing together to form mature muscle fibers when injected intoalready-developed muscle tissue, the technique of myoblast transfer hasbeen proposed as a potential therapy or cure for muscular diseases.Myoblast transfer involves injecting myoblast cells into the muscle of apatient requiring treatment. Although developed muscle fibers are notregenerative, the myoblasts are capable of a limited amount ofproliferation, thus increasing the number of muscle cells at thelocation of myoblast infusion. Myoblasts so transferred into maturemuscle tissue will proliferate and differentiate into mature musclefibers. This process involves the fusion of these mononucleated myogeniccells (myoblasts) to form a multinucleated syncytium (myofiber ormyotube). Thus, muscle tissue which has been compromised either bydisease or trauma may be supplemented by the transfer of myogenicprogenitor cells, i.e., myoblasts, into the compromised tissue.

Myoblast transfer may also be used in gene therapy, a utility enhancedby the ability of myoblasts to proliferate and fuse. Potentially,myoblasts could be genetically altered by one of several means tocomprise functional genes which may be defective or lacking in a patientrequiring such therapy. The recombinant myoblast can then be transferredto a patient, wherein they will multiply and fuse and, additionally,express recombinant genes. Using this technique, a missing or defectivegene in a patient's muscular system may be supplemented or replaced byinfusion of genetically altered myoblasts.

It has been shown that myoblasts injected into genetically deficient mdxmice fuse into the muscle fibers of the host, and are capable ofexpressing a recombinant gene product, dystrophin (an intracellularprotein, the lack of which causes Duchenne muscular dystrophy (DMD)).Partridge et al., Nature, 337: 176 (1989); Morgan et al., J. Cell.Biol., 111: 2437 (1990); Karpati et al., Am. J. Pathol., 135: 27 (1989).In a recent study involving human patients, normal myoblasts fromfathers or unaffected siblings have been transplanted into the musclesof several boys afflicted with DMD, resulting in expression of normaldonor dystrophin in the injected muscle tissue. Gussoni et al., Nature,356: 435-438 (1992). Long-term expression of a non-muscle gene product(human growth hormone) has also been achieved using myoblast transfer ofgenetically engineering myoblasts into mouse muscle. Dhawan et al.,Science, 254: 1509-12 (1991). Therefore, gene therapy using myoblasttransfer may be applied in providing essential gene products not only tomuscle tissue, but through secretion from muscle tissue to thebloodstream as well.

Although gene therapy via myoblast transfer has great potential utility,that utility is limited by the fact that, currently, there are nomyoblast-specific promoters or enhancers available to induce geneexpression in recombinant myoblasts. Such transcription control elementsare needed for myoblast-mediated gene therapy for two reasons: (1) toenable useful genes (e.g., genes involved in autocrine regulation ofmyoblast development) to be expressed in myoblasts; and (2) to restrictsuch recombinant gene expression to myoblasts and their progeny. Thus,to facilitate myoblast-mediated gene therapy, myoblast-specifictranscription control elements are needed.

Myoblast-specific enhancers of gene expression could also provide amuch-needed alternative to artificial manipulation of muscle mass inagricultural animals. Currently, muscle weight in food animals, such ascows, pigs and chickens, is manipulated by traditional breeding programsand by hormone treatment, e.g., growth hormone. Hormone treatment ofanimals to facilitate weight gain is expensive, leading to an increasedmarket price of the animal, as well as presenting potential dangers toconsumers who may be sensitive to such food additives. Clearly, ifweight gain could be mediated by an alternative means, the potentiallyhazardous use of hormone treatment could be obviated.

The availability of techniques for creating transgenic animals byintroducing inheritable genetic alterations at the embryo stage offers apotential vehicle for manipulating muscular development by geneticengineering. However, specific manipulation of progenitor embryo cellsof myogenic lineage requires the availability of myogeniclineage-specific promoters and enhancers. Otherwise, muscle-specificgenetic alterations could not be introduced. The availability of suchenhancers is necessary for the development of genetically-basedimprovements in muscle size and growth, heretofore achievable onlythrough more time-consuming or otherwise undesirable techniques, such ashormone treatment.

SUMMARY OF THE INVENTION

In accordance with the present invention, transcription control elementsthat regulate gene expression in myogenic cells are provided. Accordingto one aspect of the invention, there is provided an isolated DNAsegment having an enhancer activity in cultured cells and innon-cultured, myogenic cells. This enhancer activity causes increasedexpression of a target gene when the DNA segment and the target gene aredisposed within a DNA strand and the DNA segment is so position in the5' direction relative to the target gene to permit the increasedexpression of that target gene. In a preferred embodiment, the isolatedDNA segment having enhancer activity is isolated from a 50-100-kb regionadjacent in the 5' direction to a gene encoding a bHLH myogenicregulatory protein.

According to another aspect of the present invention, there is provideda DNA segment isolated and purified from an approximately 25.5-kbfragment adjacent in the 5' direction to a human myoD gene, having anucleotide sequence substantially the same as Sequence I.D. No. 2,described herein. In a preferred embodiment, there is provided a DNAsegment consisting essentially of a nucleotide sequence substantiallythe same as bases 1-258 of Sequence I.D. No. 2, described herein. Thereis also provided a DNA segment isolated and purified from anapproximately 18-kb fragment adjacent in the 5' direction to a quailqmf1 gene, having a nucleotide sequence substantially the same asSequence I.D. No. 3, described herein.

According to another aspect of the present invention, vectors areprovided which comprise the DNA segments described above. Additionally,procaryotic or eucaryotic host cells transformed or transfected withsuch vectors are also provided.

According to another aspect of the present invention, there are providedantisense oligonucleotides having sequences capable of hybridizing witha DNA segment having the above-described enhancer activity. Suchantisense oligonucleotides will be useful for identifying and locatingparticularly functional regions in the transcription control elements ofthe invention.

According to another aspect of the present invention, there is providedan isolated DNA segment having an enhancer activity in cultured cells,which may be isolated by a method comprising: (1) obtaining testsegments of DNA sequences from the 5' upstream region within 100-kb of agene encoding a bHLH myogenic regulatory protein, which are suspected ofhaving such enhancer activity; (2) preparing a set of test constructs,each one containing one of the test segments, a reporter gene and avector adapted for expression in a cultured eucaryotic cell, the testsegment and the reporter gene being so located relative to each otherand to any regulatory sequences of the vector to permit expression ofthe reporter gene, as well as the enhancing activity, if present, of thetest segment; (3) similarly preparing a control construct comprising areporter gene and the vector, but not having a test segment; (4)introducing the test constructs or the control constructs into culturedeucaryotic cells under conditions permitting the expression of thereporter gene (the expression of the reporter gene causes formation of adetectable product, which is formed in an amount correlatable toexpression of the gene); (5) comparing the amount of detectable productformed from the test construct with the amount of detectable productformed in cells having the control construct, the magnitude of the ratiobetween the two being indicative of enhancer activity suspected of beingpossessed by the test segment; and (6) identifying and isolating eachtest segment found to possess such enhancer activity.

According to yet another aspect of the present invention, there isprovided an isolated DNA segment having enhancer activity specificallyin myogenic cells of a living animal, which is isolated by a methodcomprising: (1) obtaining test segments of DNA sequences from the 5'upstream region within 100-kb of a gene encoding a bHLH myogenicregulatory protein, which are suspected of having such enhanceractivity; (2) preparing a set of test constructs, each test constructcomprising a test segment and a reporter gene, as well as any regulatorysequences necessary for expression of the reporter gene in cells of avertebrate embryo, all sequences being so located relative to each otherto permit expression of the reporter gene, as well as the enhancingactivity, if present, of the test segment; (3) introducing the testconstructs or the control constructs into cultured eucaryotic cellsunder conditions permitting the expression of the reporter gene (theexpression of the reporter gene causes formation of a detectableproduct, which is formed in an amount correlatable to expression of thegene); (4) determining which, if any, cells of the vertebrate embryoform the detectable product, the formation of detectable productspecifically in myogenic cells being indicative of enhancer activity;and (5) identifying and isolating the test segments possessing suchenhancer activity.

The myoblast-specific transcription control elements of the presentinvention will enable significant advances in the field of gene therapyusing myoblast transfer and microinjection techniques. Currently,genetic manipulations using these techniques are performed withrecombinant genes under the control of promoters and enhancers that arenot specificto myogenic cells. Such lack of myoblast specificity limitsthe utility of these methods for gene therapy, or other geneticengineering techniques, which requires that genes be expressed only inmyogenic cell lineages, or during the course of muscle development. Thetranscription control elements of the present invention provide therequisite myoblast specificity.

Another advantageous application of a myoblast-specific transcriptioncontrol element of the invention relates to the observation that,because an enhancer such as the myoD enhancer, is turned "on" and "off",it must itself be regulated by trancription factors operating very earlyin myogenic lineage determination. The transcription control element ofthe invention could be utilized to great advantage in biochemical assaysfor activity of such early transcription factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a map of human cosmid clone chMD-13. EcoR1 (unmarked verticallines) and NotI restriction sites, and presumed Mbo1 cloning sites areindicated. Exons and introns in MyoD are shown by solid and open blocks,respectively. For reference, restriction fragments for enzymes shown arelabelled sequentially from the 5' to the 3' end of the cosmid cloningvector. Thick line, human sequences (to scale); thin line, pWE15 vectorsequence. Numerals 1-10 beneath the thick line refer to EcoR1 fragmentscomprising chMD-13.

FIG. 1B is a map of EcoR1 Fragment 3 of cosmid clone chMD-13. Apa1,BamH1, Kpn1 and Pst1 restriction sites are shown. Numerals 0-4 beneaththe horizontal line refer to length in kilobases. The location of a 1757bp region of chMD-13 Fragment 3, corresponding to Sequence I.D. No. 2 isindicated.

FIG. 2 illustrates CAT constructs comprising upstream myoD transcriptioncontrol elements. EcoR1 restriction sites (unmarked vertical lines) areindicated. -2.5CAT and -24CAT refer to CAT reporter gene constructs withthe minimal and maximal amounts of human myoD upstream sequencesappended to the reporter gene. Thick line, human sequences (to scale);thin line, pBluescript vector sequence; dotted line, ptkCAT EH vectorsequence. CAT structural gene sequences and SV40 sequences areindicated.

FIG. 3 shows transient transfection of 23A2 myoblasts with CAT reportergene constructs containing 5' flanking sequences upstream of the humanmyoD gene. PoCAT is a promoterless CAT gene construct, and -24ΔF3CAT isidentical to -24CAT except that Fragment 3 has been deleted. Y-axis: CATactivity represented as the percent conversion of ³ H-chloramphenicol tobutyryl-³ H-chloramphenicol per microgram of protein per hour at 37° C.

FIG. 4 is a map of λ EMBL3 clone gc1120, comprising the qmf1 (QmyoD) DNAlocus from quail. EcoR1 () and Pst1 (□) restriction sites areindicated. Exons (E1, E2 and E3) of qmf1 are indicated. For reference,restriction fragments for enzymes shown are labelled sequentially fromthe 5' to the 3' end of the cloning vector (EcoRI fragments=R1-R9, PstIfragments=P1-P4).

FIG. 5 shows transient transfection of quail primary myoblasts with CATreporter gene constructs containing 5' flanking sequences upstream ofthe qmf1 gene. The CAT reporter gene was linked to the thymidine kinase(TK) promoter (pTKCatΔEH) and to the qmf1 restriction fragments shown inFIG. 4. On the x-axis, TK=thymidine kinase promoter alone; R1-R5 andP1-P4=thymidine kinase promoter combined with the restriction fragmentindicated. Y-axis: CAT activity represented as the percent conversion of³ H-chloramphenicol to butyrl-³ H-chloramphenicol per 10 μg protein perhour at 37° C.

DETAILED DESCRIPTION

The following words and phrases are defined, for reference in describingthe invention, as follows:

1. Transcription control element: refers to an isolated DNA segmentthat, under specified conditions, possesses a transcription-controllingactivity with respect to expression of a target gene. An enhancer is atype of transcription control element. Enhancers generally increase theexpression of a target gene when placed in appropriate proximitythereto. The term "transcription control element" and "enhancer" areused interchangeably herein when referring to the isolated DNA segmentsof the present invention.

2. Myogenic cell: refers to a stably determined cell type, capable ofextensive cell division, the progeny of which faithfully inherit theirmyoblast identity and possess the potential for differentiating intomature muscle fibers. The terms "myoblast" and "myogenic cell" are usedinterchangeably herein.

3. Target gene: refers to a gene upon which a transcription controlelement of the invention exerts its transcription control activity.Specifically, an enhancer element of the invention, when placed upstreamfrom the target gene causes increased expression of the target gene. Asused herein, "target genes" contain promoters. These promoters can bethe homologous promoter of the target gene or they can be a heterologouspromoter. However, the target gene must be under the control of apromoter.

4. "Substantially the same as": when referring to specific DNA sequencesset forth herein, "substantially the same as" means taking into accountminor variations or substitutions that arise for a number of reasons,but do not alter the overall characteristics of the DNA molecule definedby the sequence. For example, homologous regions isolated from differentstrains or sub-species of an animal may possess sequence polymorphismsthat render those sequences substantially the same as, but not identicalto, the sequences set forth herein. Additionally, errors in analyzingDNA sequence information, or entering such information into a recordsystem, may also produce sequences that are substantially the same, butnot identical to, the sequences set forth herein.

5. "Approximately": when used herein in describing DNA fragment lengthsor, "approximately" means within a margin of commonly acceptable errorfor the determination of DNA fragment size or relative position on a DNAstrand by standard methods, such as agarose gel electrophoresis andcomparison with standard fragments of known size.

In accordance with the present invention, it has now been discoveredthat expression of the human myoD gene is regulated not only by apromoter, but also by a distal enhancer sequence 18-22 kilobasesupstream (5') from the myoD gene. Transcriptional activity of the myoDpromoter and enhancer was assayed in myogenic cells derived from themultipotential 10T1/2 cell line by 5-azacytidine treatment. The myoDenhancer and promoter were active in myogenic and nonmyogenic celllines. The myoD promoter itself was found to be only weakly activeunless coupled with the upstream enhancer sequence. Moreover, the myoDenhancer sequence was also found to be capable of enhancing geneexpression even when coupled with a heterologous promoter, e.g., theherpes virus thymidine kinase (HSVtk) promoter.

A second myoD enhancer (referred to herein as qmf1) encoding a quailMyoD protein (QMyoD), has also been isolated and cloned. Like the humanmyoD enhancer, the qmf1 enhancer is active in myogenic and non-myogeniccell lines. The qmf1 enhancer sequence can also enhance gene expressionwhen coupled with either the qmf1 promoter, or a heterologous promoter,such as the herpes virus thymidine kinase (HSVtk) promoter. However,unlike the human myoD enhancer, the qmf1 enhancer is locatedapproximately 11.5-15 kb upstream from the qmf1 gene. Moreover, the qmf1enhancer contains no extensive sequence homology with the human myoDenhancer sequence.

It has further been found that, although the two myoD promoters andenhancers are active in non-myogenic cultured cells, in transgenic mouseembryos the human myoD enhancer and the qmf1 enhancer direct expressionof genes specifically to the skeletal muscle lineage (myoblasts) only.However, the spatial and temporal expression of genes under the controlof the human myoD enhancer is different from that of genes under thecontrol of the qmf1enhancer, as described in greater detail in Example 6below.

Insofar as is known, the above-described enhancers are the firstmyoblast-specific enhancers to be isolated and cloned. These enhancersmay be utilized with a homologous or heterologous promoter to enhancemyoblast-specific gene expression, as will be described in furtherdetail below.

The description which follows sets forth the general procedures involvedin practicing the present invention. To the extent that specificmaterials are mentioned, it is merely for purposes of illustration andis not intended to limit the invention.

I. Preparation of Myoblast-Specific Transcription Control Elements

Myoblast-specific transcription control elements may be prepared by twogeneral methods: (1) they may be synthesized from appropriate nucleotidetriphosphates, or (2) they may be isolated and purified from biologicalsources. Both methods utilize protocols that are known in the art.Unless otherwise specified, standard cloning and recombinant DNAprocedures, such as those described in Sambrook et al., MolecularCloning, Cold Spring, Harbor Laboratory (1989) (hereinafter "Sambrook etal.") are used.

Where DNA sequence information is known, a myoblast-specific enhancer ofthe invention may be prepared by oligonucleotide synthesis. Syntheticoligonucleotides may be prepared by the phosphoramadite method employedin the applied Biosystems 380A DNA Synthesizer or similar devices. Theresultant construct may be purified according to procedures well knownin the art, e.g., by high performance liquid chromatography (HPLC).Long, double-stranded polynucleotides, such as those of the presentinvention, will have to be synthesized in stages, due to the sizelimitations inherent in current oligonucleotide synthetic methods. Thus,for example, a 4-kb double-stranded DNA molecule may be synthesized asseveral smaller segments of appropriate complementarity. Complementarysegments thus produced may be annealed such that each segment possessesappropriate cohesive termini for attachment of an adjacent segment.Adjacent segments may be ligated via annealing of cohesive termini inthe presence of DNA ligase, to construct an entire 4-kb double-strandedmolecule. A synthetic DNA molecule so constructed may then be cloned andamplified in an appropriate vector.

The constructs of the present invention may be maintained in anyconvenient cloning vector. In a preferred embodiment, large constructsare maintained in a cosmid cloning/transfer vector, such as pWE15(Stratagene), which is propagated in a suitable E. coli host cell, e.g.,E. coli strain NM554 (Stratagene), and which also may be transferred tomammalian cells. Alternatively, constructs may be maintained in lambdavectors (e.g., λEMBL), which are often used to construct genomiclibraries. Smaller constructs are conveniently cloned into plasmids.

Myoblast-specific transcription control elements may be isolated fromappropriate biological sources using methods known in the art. In onepreferred embodiment, a human myoD transcription control element isisolated and cloned. A myoblast genomic library may be constructed incosmid clones. The library can be screened with a cDNA, such as a fulllength mouse myoD cDNA as described by Pinney et al., Cell, 53: 781(1988). Clones identified by such a screening may be analyzed, e.g., byrestriction mapping, for the presence of significant amounts of upstreamDNA sequence. Clones having up to 50 kb 5' to the myoD gene may beselected and analyzed for enhancer and promoter activity. Due tolimitations in current technology, commonly-used cloning vectors cannotcontain more than approximately 50 kb of inserted DNA. For this reason,the 5' region 50-100 kb from the myoD gene will have to be identified bya second screening step, using previously-identified clone containingupstream sequences 25-50 kb from the myoD gene. Such strategies forobtaining far distal sequences are commonly employed by those skilled inthe art.

A cosmid clone comprising the human myoD gene and an approximately 25 kbupstream region, which constitutes a myoblast-specific transcriptioncontrol element in accordance with the present invention, wasconstructed. A diagram of this construct is provided in FIG. 1A. Thehuman myoD transcriptional control element comprises several distinctivefeatures. A promoter region is present in the 2.5 kb fragmentimmediately 5' to the gene. The DNA sequence of the human myoD gene,including 1 kb of upstream sequence comprising the promoter region, isset forth below, as Sequence ID No. 1. The locations of the putativeTATA box, the 3' end of the promoter region, and the translation startsite of the myoD gene product are indicated. ##STR1##

An enhancer element is present in a 4 kb fragment approximately 18-22 kbupstream from the human myoD gene (Fragment 3). A restriction map ofthis fragment is shown in FIG. 1. The DNA sequence of approximately 1.7kb of this fragment is set forth hereinbelow as Sequence ID No. 2.##STR2##

The two subregions of the sequenced portion of the enhancer elementidentified to date which have particular enhancing activity map to bp1-258 and 1185-1757. The subregion mapping to bp 1-258 appears to confermyoblast specificity, while the 1185-1757 subregion affects the amountof expression-enhancing activity. It will be apparent to those skilledin the art that other regions, particularly 5' to the sequenced regionmay also have activity.

In another preferred embodiment, a quail myoD transcription controlelement is isolated and cloned. In this embodiment, a genomic libraryfrom quail embryonic primary myofibers is constructed in a lambdavector, and screening with a cDNA encoding a QMyoD regulatory protein,such as the quail qmf1 cDNA clone as described by de la Brousse et al.,Genes and Devel., 4: 567-581 (1990). Clones identified by the screeningare analyzed for the presence of significant amounts of upstream DNAsequences, and clones having up to 50 kb 5' to the qmf1 are selected andanalyzed for enhancer and promoter activity. DNA sequences fartherupstream may also be analyzed by conducting a secondary screening asdescribed above.

A λ EMBL3 clone comprising the qmf1 gene and approximately 18 kbupstream region, which constitutes a myoblast-specific transcriptioncontrol element in accordance with the invention, was isolated. Adiagram of this construct (referred to as gc1120) is provided in FIG. 4.

An enhancer element is present in a 2.2 kb fragment approximately11.5-15 kb upstream from the qmf1 gene, in restriction fragment P3. TheDNA sequence of fragment P3 is set forth hereinbelow as Sequence I.D.No. 3. ##STR3##

It is commonly expected that expression of a gene will be controlled atleast in part by a promoter sequence situated immediately 5' to thetranscription start site. However, some gene are additionally regulatedby enhancer elements, which can be located at positions far removed fromthe gene itself. Such enhancer elements may be found far upstream fromthe gene (as much as 50-100 kb, in some instances), or downstream fromthe gene in the 3' untranslated region, or even within the gene itself,in an intron. Alternatively, a gene may be expressed without the controlof any enhancer element. See S. D. Gillies et al., Cell 33: 717-728(1983); E. Serfling et al., Trends in Genet. 1: 224-230 (1985).

In spite of the difficulty in predicting if, or where, an enhancerelement may exist, once an enhancer sequence for a particular gene isidentified, it is likely that a similarly situated enhancer element willalso be presented in related genes, or homologous genes from otherspecies. Thus, in accordance with the present invention, theabove-described human myoD enhancer was discovered and characterized asa 1.7 kb fragment existing 18-22 kb upstream from the human myoDtranscription start site. Once the human myoD enhancer had beendiscovered and located, according to methods described herein, theupstream region of the quail myoD gene (qmf1) was examined for thepresence of a similarly situated enhancer element. Such an enhancer wasidentified, at approximately 15-17 kb upstream from the qmf1transcription start site. Although this enhancer is of differentsequence homology from the human myoD enhancer, and directs a somewhatdifferent pattern of myogenic development in mouse embryos, it comprisesthe basic characteristics of the myoblast-specific enhancer elementprovided by the present invention.

Thus, both the human myoD gene and the quail qmf1 gene have been shownto possess upstream enhancers of gene expression. As described in theBackground section, MyoD and the qmf proteins are part of the bHLHfamily of myogenic regulatory proteins. These proteins have been shownto possess a high degree of evolutionary conservation. Pownall et al.,supra. For this reason, the presence of an upstream enhancer in both thehuman myoD gene and the qual qmf1 gene is a clear indication that suchan enhancer element is also present in the other genes of the bHLHmyogenic regulatory protein family. This is even further the case whenit is observed that human myoD and quail qmf1 are not the most closelyrelated members of the bHLH family.

In view of the relationship among the members of the BHLH myogenicregulatory protein family, this invention provides a transcriptioncontrol element which comprises an upstream enhancer from any one of thebHLH family. The bHLH family includes, but is not limited to: (1) MRF4(mouse, rat, human); (2) myog (chick, mouse, rat, human); (3) MyoD(human, sheep, mouse, Xenopus, Drosophila, sea urchin, C. elegans, quail(including qmf1, qmf2 and qmf3); (5) myogenin; and (6) myf5 (bovine,human, Xenopus). The methods set forth herein for analyzing the upstreamregion of any bHLH myogenic regulatory gene will be appropriate foridentifying and locating such enhancer elements. Moreover, it will beapparent to one skilled in the art that the preferred way foridentifying such an enhancer is through a functional assay, as describedherein. For example, the two myoD enhancer elements specificallyexemplified herein both possess the same basic functionalcharacteristics of enhancing gene expression in non-myogenic or myogeniccultured cells, and specifically enhancing gene expression innon-cultured myoblasts, even though the respective enhancers do notexhibit sequence homology.

Myoblast-specific transcription control activity may be analyzed bypreparing constructs in which the putative enhancer element ispositioned upstream from a common reporter gene, such as the geneencoding chloramphenicol acetyltransferase (CAT). Methods for testingthe promoter/enhancer activity of cloned DNA segments using the CATreporter gene are described in greater detail in the examples below.

Once enhancer sequences have been identified, their myoblast-specificitymay be tested in vivo by examining reporter gene expression intransgenic mouse embryos. The transcription control element is coupledto a reporter gene, such as the lacZ gene, and introduced into animalembryos by pronucleus injection, according to known methods. Reportergene expression may be monitored by observing whole mounts and seriallysectioned embryos several days (e.g., 11-12) post-coitum, the time atwhich myogenic cells of the somatic myotome limb buds first expressmyogenic gene transcripts at high concentration. If the putativetranscription control element is indeed myoblast-specific, and active inmyogenic cells, the reporter gene should be expressed under theseconditions. Methods of testing potential myoblast-specific transcriptioncontrol elements in vivo are described in greater detail in the examplesbelow.

II. Methods of Using Myoblast-Specific Transcription Control Elements

A. Somatic Gene Therapy

The transcription control elements of the present invention may be usedto considerable advantage in myoblast-mediated gene therapy. Becausemyoblasts proliferate and fuse together, they are capable ofcontributing progeny comprising recombinant genes to multiple,multinucleated myofibers in the course of normal muscular development.Dhawan et al., Science, 254: 1509-12 (1991). The transcription controlelement of the present invention may be used in conjunction withexisting myoblast transfer techniques to provide high expression ofrecombinant genes, as well as great specificity of gene expression. Itshould be noted that a significant feature of the transcription controlelement of the present invention is that it is inactive after myoblastshave differentiated into mature muscle cells. Thus, such an elementprovides a needed control of gene expression, whereby recombinant genesmay be expressed during myoblast proliferation of fusion, but will be"turned off" once the myoblast cells and their progeny havedifferentiated, or shortly thereafter. In a preferred embodiment, thetranscription control element of the invention comprises an enhancerelement that becomes inactive once myoblasts have differentiated intomuscle cells, used in conjunction with a promoter element that is notmyoblast-specific.

Myoblast transfer, using genetically engineered myoblasts according tothe present invention, may be accomplished by methods known in the art.See, e.g., Dhawan et al., supra. For example, cultured myoblast cellsmay be genetically altered to comprise stably incorporated recombinantgenes under myoblast-specific transcriptional control using transfectionor infection methods known in the art. Such transfection or infectionmethods include transfection via mammalian expression vectors orhigh-efficiency retroviral-mediated infection. Myoblasts geneticallyaltered in this way are then examined for expression of the recombinantgene. Such genetically altered cells are expanded for introduction intomuscle tissue in vivo.

For injection of myoblasts into muscle, cells may be trypsinized, washedand suspended in, e.g., phosphate buffered saline (PBS). 10⁶ -10⁷myoblasts may be delivered in a small volume (e.g., 10-100 microliters)in a series of several injections throughout the muscle tissue to betreated. The transferred recombinant myoblasts will express recombinantgene product during the period in which they proliferate and begin tofuse with existing muscle cells. However, once this period ends, theenhancer controlling the recombinant gene will be deactivated, whereuponrecombinant gene expression ceases.

Myoblast transfer using a myoblast-specific enhancer of the presentinvention may be employed to particular advantage in manipulatingautocrine regulation of muscle development (i.e., the ability of amyoblast to regulate its own growth). Genes encoding growth factors(e.g., FGF or insulin-like growth factors), placed under the control ofa myoblast-specific enhancer of the invention, could be used togenetically alter myoblasts. These myoblasts could be transferred intomuscle, where the recombinant genes would be expressed. The growthfactors expressed by the recombinant myoblasts would enable therecombinant cells to proliferate as myoblasts to a greater extent thanwould a non-recombinant myoblast, thus expanding the population ofmyogenic cells in the muscle tissue being treated.

Constructs containing a potentially useful gene under the control ofmyoblast-specific transcription control elements of the invention may betested in cultured cells prior to undertaking myoblast transfer into aliving organism. For example, such a construct, placed in an appropriateexpression vector, may be used to transfect 23A2 myoblasts, as describedin greater detail in Example 2 below. The amount of recombinant proteinexpressed by the contruct may be measured according to standard methods(e.g., by immunoprecipitation). In this manner, it can be determined invitro whether a potentially useful protein, encoded by a gene under thecontrol of a transcription control element of the invention, is capableof expression to a suitable level that it will be appropriate formyoblast transfer.

Additionally, potentially useful constructs comprising recombinant genesunder the control of transcription control elements of the invention maybe tested in developing animal embryos. Testing of expression inembryonic animals may more closely approximate expression conditions inmyoblasts used for myoblast transfer. Methods for introducing suchconstructs into mouse embryos are described in greater detail in Example3 below.

B. Germline Genetic Manipulation

As mentioned earlier, recombinant genes under the control oftranscription control elements of the present invention may be used forgenetic alteration of embryonic cells to create transgenic animals withimproved muscular characteristics. A recombinant gene under the controlof a myoblast-specific transcription control element may be introducedby pronucleus injection, as described in Example 3 below. Instead ofsacrificing the embryos, as described in the Example, the embryos may beimplanted into a recipient female and the animals allowed to be born.Putative transgenic animals can be raised and then bred to determine ifthere has been inheritable incorporation of the recombinant gene intothe animal's genome.

During embryological development of a transgenic animal, the recombinantgene should remain dormant until such time as myoblast determinationbegins. The recombinant gene should then become activated in themyoblasts, conferring the benefit of the selected recombinant geneproduct. For example, a gene encoding growth hormone may be placed underthe control of the transcription control element of the invention, andbecome activated throughout the period of the animal's growth in whichmuscle formation is occurring. As muscle tissue matures, thetranscription control element will become deactivated, as mentionedearlier, and the recombinant gene product will cease to be produced.

It should be noted in this regard that the mouse embryological systemdescribed hereinabove, and in Example 3 below, is an extremely usefulanimal model system for testing recombinant genes under the control oftranscription control elements of the invention. These animals may bemanipulated in the laboratory as embryos, and also raised to adulthoodunder controlled conditions. Thus, potentially useful recombinant genesmay be screened for expression and effectiveness throughout the growthperiod of a mouse, and evaluated on that basis for efficacy in otheranimals.

The following examples are provided to describe the invention in furtherdetail. These examples are intended to illustrate and not to limit theinvention.

EXAMPLE 1 Isolation and Cloning of a Human myoD Transcription ControlElement

The transcription control element that regulates expression of the humanmyoD gene was identified and cloned. A full-length mouse myoD cDNA(Pinney et al., Cell, 53: 781, 1988) was used to screen a pWE15 humangenomic MboI cosmid library (Stratagene, La Jolla, Calif.).Approximately 400,000 colonies on 20 duplicate nitrocellulose filterswere hybridized at moderate stringency (65° C. for pre-hybridization andhybridization, 55° C. for washes) with a ³² P-labelled random-primedmouse MyoD1 cDNA.

This screen yielded 4 recombinants representing 3 unique overlappingclones that spanned a total of 40 kb. Sequence comparison with humanMyoD cDNA identified the hybridizing species as myoD.

EcoR1 maps of the clones were generated by the indirect end-labellingmethod, as described by Wahl et al., Proc. Nat'l. Acad. Sci. (USA), 84:2160 (1987). The organization of the cosmid clone used in subsequentanalysis (chMD-13) includes approximately 25.5 kb of DNA upstream and 4kb downstream of the myoD gene, as shown in FIG. 1A. The approximatesizes of the restriction fragments of chMD-13 are as follows: 1, 1.7 kb;2, 1.9 kb; 3, 4.1 kb; 4, 0.45 kb; 5, 9.7 kb; 6, 0.65 kb; 7, 0.25 kb; 8,3.9 kb; 9, 6.4 kb; 10, 2.8 kb.

EXAMPLE 2 Measurement of Transcription Control Activity of amyoD-Regulating Transcription Control Element in Cultured Myogenic Cells

Transcriptional activity of the myoD transcription control elementdescribed in Example 1 was assayed in 23A2 myoblasts, myogenic cellsderived from the multipotential 10T1/2 cell line by 5-azacytidinetreatment. Konieczny et al., Cell, 38: 791 (1984). This was accomplishedby constructing several clones wherein different regions of the flankingsequence of myoD were fused to the chloramphenicol acetyltransferase(CAT) reporter gens, then assaying for CAT activity after transienttransfection into proliferative 23A2 myoblasts.

All cell lines were obtained from the American Type Culture Collectionexcept 23A2, which was derived from 10T1/2 cells by 5-azacytidinetreatment, as described above. C3H10T1/2 and 23A2 cells were maintainedin Basal Medium Eagle (BME) medium supplemented with 15% fetal bovineserum (FBS). JEG-3 human choriocarcinoma cells were maintained inDulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, HepG2human hepatoma cells were maintained in 50:50 DMEM Ham's F12supplemented with 10% FBS. All media was supplemented with penicillin G(100 U/ml) and streptomycin sulfate (100 μg/ml) (Gibco, Grand Island,N.Y.). All DNAs used in transfections were prepared by alkaline lysisand double banded in CsCl gradients, according to standard methods.Cells were transfected by the calcium phosphate precipitation method asfollows. Cells were trypsinized and plated at 2×10⁵ cells per 100-mmplate (10T1/2 and 23A2 cells) or passed ˜1:10 from 50% confluent plates(JEG-3 and HepG2 cells). The following day cells were fed fresh medium,and 3 hours later calcium phosphate-DNA coprecipitates (1 ml per 100-mmdish) were added (0.8, pmole of test vector, brought to 25 μg withvector carrier DNA). About 16 to 18 hours later, the precipitates wereremoved, cells washed one time in basal medium without FBS, and then fedcomplete medium. After 48 hours, cells were harvested and lysed byfreeze-thawing. CAT enzyme activity in cell extracts was quantified withthe xylene extraction method, as described by Seed et al., Gene, 67: 271(1988), with ³ H-labeled chloramphenicol (31.2 Ci/mmole, New EnglandNuclear) and N-butyryl coenzyme A (Sigma, St. Louis, Mo.). Equivalentamounts of protein (15 to 25 μg as determined with a BioRad protein kitand bovine serum albumin as a standard) and a reaction time of 1 hourwere used in all CAT assays, which kept all values within the linearrange of the assay. In a typical experiment with 15 μg of protein, 1%conversion of ³ H-labelled chloramphenicol to butyryl ³H-chloramphenicol was ˜45,000 cpm as determined by scintillationcounting.

The CAT constructs are shown in FIG. 2. The constructs were prepared bythe following method. ptkCATΔEH, derived from pBLCAT2 (Luckow et al.,Nuc. Acids Research, 15: 5490 (1987)), by deletion of the Nde 1-Hind IIIfragment of pUC 18, was used in all transfection experiments. SimilarCAT activity vectors are also commercially available from, e.g., PromegaBiotech (Madison, Wis.), and may be substituted for the vectors usedherein. All cloning procedures were by standard methods. A 2.8 kbfragment containing the myoD promoter, derived from pBluescript II KS+(a widely pUC derivative available from Promega Biotech) sequencingdeletion, was generated by digestion with SacI followed by partialdigestion with Kpn I (both sites derive from the multiple cloning siteof the pBluescript vector) and was blunt-end-ligated into ptkCATΔEHafter digestion with Xba I and Bgl II (thereby removing all HSVtkpromoter sequences (from -105 to +51). The resulting construct contains˜2.7 kb of human sequences extending from an Eco R1 site ˜2.5 kb 5' ofthe myoD gene (see FIG. 1A) to +198 relative to the TATA box (nucleotide-37 relative to the start of translation; see Sequence I.D. No. 1). The-24CAT construct was generated by digesting chMD-13 with NotI, followedby partial cleavage with EcoR1. Partial cleavage products weresize-fractionated on a 0.6% agarose gel, and fragments of about 20 to 25kb were gel purified and directionally cloned into -2.5CAT that had beendigested with NotI and partially digested with EcoR1 (vector sequencesin -2.5CAT contain two EcoR1 sites). The resulting clone containedcontinuous human sequences from the distal NotI site through +198.Fragments 2 through 8 were cloned into the Xba1 site of -2.5CAT bydigesting chMD-13 with NotI and EcoR1, and blunt end-ligating fragmentsinto the unique Xba1 site (see FIG. 2). Fragment 3 was cloned in bothorientations upstream of the tk promoter by blunt-end-ligation into theunique BamH1 site (F3/tkCAT and F3'/tkCAT). The -24ΔF3CAT construct wasgenerated by partially digesting -24CAT with EcoR1 ligatinggel-purified, size-selecting digestion products, and screening by colonyhybridization for clones missing only fragment 3.

As shown in FIG. 2, -2.5CAT and -24CAT refer to CAT reporter geneconstructs with the minimal and maximal amounts of human myoD 5'sequences tested in transient transfection assays. These sequences in-2.5CAT extend from the EcoR1 site ˜2.5 kb upstream of myoD to +198relative to the TATA box (see Sequence ID No. 1). Fragments 2 through 8of chMD-13 (see FIG. 1A) were tested for transcriptional enhancingactivity after cloning into the XbaI site of -2.5CAT, as shown in FIG.2.

The results of transient transfection assays are shown in FIG. 3. It canbe seen that the construct comprising the myoD promoter region (-2.5CAT;FIG. 2) yielded CAT activity 5-10 fold greater than a promoterless CATconstruct (PoCAT). The -2.5CAT activity constituted ˜20% of CAT activityachieved when another promoter, the herpes virus thimidine kinase(HSVtk) promoter is used (data not shown). Moreover, addition to -2.5CATof fragments F2 or F4-F8 yielded no significant increase in CATactivity. However, when Fragment F3 was added to -2.5CAT, CAT activitywas stimulated ˜10 fold above the promoter alone. In fact, activity ofthat construct was even greater than activity of -24CAT, which comprisesall of fragments F1-F8 (FIG. 2). The F3 fragment was shown to becritical for CAT expression through the construction of a CAT constructcomprising F1-F8, but lacking F3 (-24ΔF3CAT). This construct stimulatedCAT activity no better than the myoD promoter above (FIG. 3).

Thus, the myoD enhancing activity was quantitatively recovered in afragment 18-22 kb 5' to myoD (Fragment 3, FIG. 1A). In addition toenhancing the activity of the myoD promoter, Fragment 3 was also foundto enhance the activity of the HSVtk promoter, and was equally effectivein both orientations. In addition, Fragment 3 in either orientationexhibited only background CAT activity in a promoterless CAT construct,demonstrating that Fragment 3 does not contain promoter activity.

Because myoD is expressed exclusively in skeletal muscle, the musclespecificity of the myoD transcription control element was investigated.The 10T1/2 cells are non-myogenic and do not express myoD, but areconverted to myogenic cells by 5-azacytidine, by forced expression ofthe myogenic regulatory cDNAs, and by transfection of the genomic locusmyd. The myoD promoter and enhancer, as well as the entire 24 kb of 5'flanking sequence, were as active in 10T1/2 cells as in 23A2 myoblasts.In stable transfection assays, these control elements also showedcomparable activity in 10T1/2 and 23A2 cells.

A variety of cell lines were tested to determine whether multipotential10T1/2 cells were unique among non-myogenic cells in their ability toexpress the myoD promoter and enhancer. These included Ltk- cells, three10T1/2-derived adipocyte cell lines, BNL liver cells, HepG hepatomacells and JEG-3 choriocarcinoma cells. The myoD enhancer and promoterwere active in all of these cell lines except JEG-3 cells. Activity wasrelatively low in HepG2 cells, but in the other cells lines wascomparable to that in 23A2 myoblasts. Similarly, the resident human myoDgene was activated when chromosome 11 was transferred from primary humanfibroblasts to various tissue culture cell lines. Expression of the myoDenhancer and promoter in these non-myogenic cells, which do not expressany known helix-loop-helix myogenic regulatory proteins, indicates thattheir activity is not dependent on auto- or cross-activation by membersof the helix-loop-helix myogenic protein family.

EXAMPLE 3 In Vivo Myoblast Specificity of a myoD Transcription ControlElement

Although the myoD transcription control element is active innon-myogenic cultured cells, it was found to be specific for myogeniccells in vivo. To determine this in vivo specificity, two lacZ reportergene constructs were tested in transgenic mouse embryos.

The lacZ vector, pPD46.21 was used in transgene constructions. pPD46.21is identical to pPD1.27 (Fire et al., Gene, 93: 189 (1990)) except thatit lacks the sup-7 gene. It contains an initiation codon and SV40 Tantigen nuclear localization signal just upstream from lacZ, andpolyadenylation sequences from the SV40 early region downstream of lacZ.Similar lacZ reporter constructs are commercially available (e.g.,Promega Biotech, Madison, Wis.) and may be substituted for the lacZconstructs used herein. The -2.5lacZ and F3'/-2.5lacZ vectors wereconstructed by digesting -2.5CAT and F3'/-2.5CAT at flanking SalI andXhoI sites and cloning gel-purified fragments into the SalI site in the5' polylinker of pPD46 21 (thereby destroying the XhoI site). The-2.5lacZ and F3'/-2.5lacZ vectors yielded a faint or intense, nuclearlocalized signal, respectively, after transient transfection into 23A2myoblasts (data not shown). DNAs for injection were digested with NotIto remove pUC19 sequences, and lacZ fusions were purified on agarosegels. Microinjections of the plasmid-free lacZ fusion genes into thepronuclei of fertilized eggs of the commercially available inbred strainFBV/N were performed according to standard methods. See, Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1986); M. Shani, Mol. Cell Biol., 6: 2624 (1986).Embryos 11.5 days postcoitum (p.c.) were stained for 30 to 60 min in 1%paraformaldehyde, 0.2% glutaraldehyde in 0.1M phosphate buffer, pH 7.4.After rinsing, embryos were stained for β-gal according to the method ofSanes et al., EMBO J., 5: 3133 (1986). Following photomicrography, theembryos were embedded in paraffin, serially sectioned at 8 μm, andsections were counterstained with nuclear fast red.

The promoter/lacZ construct (-2.5lacZ) contained 2.5 kb of humansequences 5' to the myoD gene cloned upstream of lacZ, whereas the other(F3'/-2.5lacZ, contained the 2.5 kb of flanking DNA as well as theupstream enhancer fragment cloned in an antisense orientation. The-2.5lacZ construct was introduced into nine embryos by pronucleusinjection. Whole mount and serially sectioned embryos were analyzed forβ-galactosidase (β-gal) activity at 11.5 days p.c., the time at whichmyogenic cells of the somitic myotome and limb buds first express myoDtranscripts at high concentrations. None of the mouse embryos injectedwith -2.5lacZ showed lacZ expression in somites, limb buds, or any otherpopulations of myogenic cells.

Four of fourteen embryos injected with F3'/-2.5lacZ containedlacZ-expressing cells. In all four embryos, this transgene was activatedin cells from every skeletal muscle-forming region shown by in situanalyses to express the endogenous myoD gene. The most prominent featureof these embryos was the intense staining of the somites and limb buds.Somite staining yielded a metameric pattern of β-gal-positive cellsalong the central axis of the embryo. Observations of histologicalsections of three embryos demonstrated that somitic lacZ staining wasconfined to cells in the myotomal compartment of the somite. At 11.5days p.c., lacZ-expressing cells were observed in the myotomes of onlythe 20 to 25 most rostral somites; lacZ expression was not detected insomites approximately at the level of, or caudal to, the hind limb. Inlater stage embryos all somites expressed the lacZ transgene. Thisclearly defined rostrocaudal gradient of lacZ expression, whichcorresponds to the gradient of transcript accumulation for myoD and theother myogenic regulatory factors, reflects the rostrocaudal sequence ofsomite formation and maturation. The lacZ transgene is likely activatedin a ventral to dorsal sequence because lacZ-expressing cells areconfined to the ventral myotome in less mature caudal somites, but arepresent throughout the ventra-dorsal myotomal axis in more matureanterior somites.

All four lacZ-positive embryos contained β-gal-expressing cells in theproximal region of both the fore- and hind-limb buds. These cells werelocalized to the dorsal and ventral premuscle masses, which give rise tothe skeletal musculature of the limb. The fore limb contained largepopulations of cells that expressed the transgene, whereas the hind limbcontained few lacZ-expressing cells. Because myoblasts of the developinglimb buds are derived from the somite dermomyotome, the smallerpopulation of lacZ-expressing cells in the hind-limb bud probablyreflects the earlier developmental stage of the somites at the level ofthe hind limb compared to rostral somites at the level of the fore limb.

The lacZ-expressing cells were also observed in the visceral arches,evident in whole mounts as patches or anteroposterior arrays of stainedcells. In histological sections, groups of stained cells were found inthe mesenchyme of the visceral arches, organized in centrally andperipherally localized masses. Transcripts for myoD, myogenin, Myf-5co-localize to these regions of the visceral arches, which is comparedof cells that will contribute to pharyngeal and facial musculature. Inaddition, presumptive muscle of the developing diaphragm stainsintensely for β-gal. The lacZ transgene was not expressed in smooth andcardiac muscle, muscle types that do not express myoD. A stabletransgenic line carrying this lacZ transgene gave the same, skeletalmuscle-specific pattern of lacZ expression. These transgenic dataestablish that the myoD enhancer and promoter, which together constitutethe myoD transcription control element, are the DNA elements throughwhich myoD expression is regulated.

EXAMPLE 4 Isolation and Cloning of a quail myoD (qmf1) transcriptioncontrol element

The enhancer element that regulates expression of the quail qmf1 genewas identified and cloned. A qmf1DNA clone (gC1083) (de la Brousse etal., supra) was used to screen a genomic DNA library of partial Mbo1restriction fragments of quail embryonic primary myofiber DNA, ligatedinto BamHI-digested lambda EMBL3 arms (Stratagene), and plated onbacterial strain LE392. A total of approximately 400,000 primary plaqueswere screened, and 2 positive clones that hybridized under highstringency conditions (65° C. for prehybridization and washes) to agenomic fragment containing only 5' upstream sequences of the qmf1 genewere isolated. One of these clones was found to be similar to thepreviously-mapped lambda Charon 4A clone (de la Brousse et al., supra)while the other was found to overlap with that clone only in the firstExon region of the qmf1 gene. Restriction mapping of this latter genomicclone, referred to as gC1120, indicated that it contained approximately18 kb of 5' qmf1 upstream region. A restriction map of GC1120 is setforth in FIG. 4. The approximate sizes of the restriction fragments (inkb) of gC1120 are as follows: R1, 6.1; R2, 2.0; R3, 4.5; R4, 1.7; R5,0.7; R6, 4.1; R7, 0.8; R8, 6.5; R9, 4.1; P1, 0.7; P2, 1.8; P3, 2.2; P4,1.4.

EXAMPLE 5 Measurement of Transcriptional Control Activity ofqmf1-Regulating Transcription Control Element in Cultured Myogenic Cells

Transcriptional activity of the qmf1 upstream region described inExample 4 was assayed according to the methods set forth in Example 1,except that quail primary myoblasts were utilized for transfection byqmf1-CAT reporter gene constructs instead of 23A2 myoblasts and a qmf1promoter was used.

For the CAT constructs, the CAT gene linked to the herpes virus TKpromoter (pTKCATΔEH) was linked to each of the qmf1 restrictionfragments shown in FIG. 4. Transcription enhancer activities of eachrestriction fragment was tested by measuring the CAT activities of cellextracts reported as percent conversion of ³ H-chloramphenicol tobutyryl-³ H-chloramphenicol per 10 μg protein per hour at 37° C.

The results of the transient transfection assays are shown in FIG. 5. Itcan be seen that the construct comprising the R1 or the P3 restrictionfragments yielded the greatest CAT activity, 12-20 times greater thanthe CAT gene linked to the TK promoter alone. As can be seen from FIG.2, the R1 region approximately 11.5-15 kb upstream from the qmf1 gene,in which is located the 2.2 kb sequence identified herein as SequenceI.D. No. 3.

EXAMPLE 6 In Vivo Myoblast Specificity of a Quail MyoD (qmf1)Transcription Control Element

To determine the in vivo specificity of the qmf1 enhancer, a lacZreporter gene construct comprising the aforementioned P3 restrictionfragment was tested in transgenic mouse embryos, according to methodsdescribed in Example 3. The P3 lacZ plasmid for use in transgenic micewas constructed by first cloning a 2.4 kb SalI/XbaI fragments containingthe qmf1 promoter into the SalI/XbaI sites of PD46.21. The 2.2 kb P3fragment was then cloned into a PstI site adjacent to the qmf1 promoterto yield the final P3-qmf1-lacZ construct. This construct was utilizedas described in Example 3.

The qmf1 enhancer was found to control lacZ expression in transgenicmouse embryos in a manner similar to, but not exactly like, thatobserved for the human myoD enhancer described in Example 3. The qmf1enhancer was found to direct expression of the lacZ reporter gene in themyotome of the rostral somites by day 9 in transgenic embryos, whereasthe human myoD enhancer directed expression later in myotomes (i.e., byDay 10-10.5). The qmf1 enhancer was expressed in the limb buds by Day12.5, which is later than the human myoD enhancer, expressed at Day10-10.5. The earlier expression of qmf1 in somite is localized to thecentral myotomal cell, and activation proceeds in a rostral-caudalprogression. The later activation of the human myoD-enhancer-controlledlacZ occurs first at the level of the forelimb bud with prominentstaining in the ventral regions of somites. Subsequent expression occursin more anterior somites, which exhibit more dorsal activation than inthe remaining somites. In contrast to the qmf1-enhanced lacZ, the humanenhancer was found not to direct expression predominantly to the centralmyotomal muscles. Thus, the qmf1 and human myoD enhancers possessdifferent developmental timing of expression in the somites and limb,and different spatial expression in the somites. These differences inspatial expression likely reflects the formation of different lineagesof myogenic cells that give rise to different muscles of the embryo.However, both qmf1 and human myoD expression is restricted to the earlyembryonic myogenic lineages of the somite.

While certain aspects of the present invention have been described andexemplified above as preferred embodiments, various other embodimentsshould be apparent to those skilled in the art from the foregoingdisclosure. The present invention, therefore, is not limited to theembodiments specifically described and exemplified above, but is capableof variation and modification without departure of the scope of theappended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4086 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACAGACTCCACAAATCACACAGTTGGAAACTCTGAGTCTGCACTCAACTGGTCTGCAAAC60                CGCACTCTCGGAGACTTCAGGTGAGATGAGGTCAGGTTCTCAGGCCAGGTCCTGAAGTTT120               GACACCTTGGCGAAATGCACTTTCCTTGACTCAGCACCGCGAGTGAGGCGGAGCCAAGCC180               CCGAGCAGAAGGGTTTTCTTCCCAGCTGAAGAGGCAGCTCAGCCTAGACCCCAGGCATGG240               CACTGGACACCCCTGCTGTGGAAACGTGCAGATTTAGATGGAGGGGATTCCTAACCTGGG300               CAGGATCCGAGTTTGGAGAGATTGGCGCGAACGTTTAGCAGCAATCTCCGATTCCTGTAC360               AACCATAGCTGGGTTTCTAAGCGTCTAGGGAAGAAGGACTGGGCCCACGACCTGCTGAGC420               AACTCCCAGGTCGGGGACTGGCGGAATATCAGAGCCTCTACGACCCGTTTGTCTCGGGCT480               CGCCCACTTCAACTCTCGGGGTCTCTCCGCCTGTTGTTGCACTCGTGCGTTTCTCTGCCC540               CTGACGCTCTAAGCTTTCTGCTTTCTGCGTGTCTCTCAGCCTCTTTCGGTCCCTCTTTCA600               CGGTCTCACTCCTCAGCTCTGTGCCCCCAATGCCTTGCCTCTCTCCAAATCTCTCACGAC660               CTGATTTCTACAGCCGCTCTACCCATGGGTCCCCCACAAATCAGGGGACAGAGGAGTATT720               GAAAGTCAGCTCAGAGGTGAGCGCGCGCACAGCGTTTCCCGCGGATACAGCAGTCGGGTG780               TTGGAGAGGTTTGGAAAGGGCGTGCCGGAGAGCCAAGTGCAGCCGCCTAGGGCTGCCGGT840               CGCTCCCTCCCTCCCTGCCCGGTAGGGGACCTAGCGCGCACGCCAGTGTGGAGGGGCGGG900               CTGGCTGGCCAGTCTGCGGGCCCCTGCGGCCACCCCGGGGACCCCCCCCAAGCCCCGCCC960               CGCAGTGTTCCTATTGGCCTCGGACTCCCCCTCCCCCAGCTGCCCGCCTGGGCTCCGGGG1020              CGTTTAGGCTACTACGGATAAATAGCCCAGGGCGCCTGGCGAGAAGCTAGGGGTGAGGAA1080              GCCCTGGGGCTGCCGCCGCTTTCCTTAACCACAAATCAGGCCGGACAGGAGAGGGAGGGG1140              TGGGGACAGTGGGTGGGCATTCAGACTGCCAGCACTTTGCTATCTACAGCCGGGGCTCCC1200              GAGCGGCAGAAAGTTCCGGCCACTCTCTGCCGCTTGGGTTGGCGAAGCCAGGACCGTGCC1260              GCGCCACCGCCAGGATATGGAGCTACTGTCGCCAACCGCTCCGCGACGTAGACTGACGGC1320              CCCCGACGGCTCTCTCTGCTCCTTTGCCACAACGGACGACTTCTATGACGACCCGTGTTT1380              CGACTCCCCGGACCTGCGCTTCTTCGAAGACCTGGACCCGCGCCTGATGCACGTGGGCGC1440              GCTCCTGAAACCCGAAGAGCACTCGCACTTCCCCGCGGCGGTGCACCCGGCCCCGGGCGC1500              ACGTGAGGACGAGCATGTGCGCGCGCCCAGCGGGCACCACCAGGCGGGCCGCTGCCTACT1560              GTGCCTGCAAGGCGTGCAAGCGCAAGACCACCAACGCCGACCGCCGCAAGGCCGCCACCA1620              TGCGCGAGCGGCGCCGCCTGAGCAAAGTAAATGAGGCCTTTGAGACACTCAAGCGCTGCA1680              CGTCGAGCAATCCAAACCAGCGGTTGCCCAAGGTGGAGATCCTGCGCAACGCCATCCGCT1740              ATATCGAGGGCCTGCAGGCTCTGCTGCGCGACCAGGACGCGCGCCCCCTGGCGCCGCAGC1800              CGGCCTTCTATGCGCCGGGCCCGCTGCCCCCGGGCCGCGGCGGCGAGCACTACAGCGGCG1860              ACTCCGACGCGTCCAGCCCGCGCTCCAACTGCTCCGACGGCATGGTAAGGCCGGGACCCC1920              AGGAAGTGAGGAAGTTAGGGCGGCGCTCGGGATATCAGGGACGCGTTTCCGAGGGCGGGG1980              AGCTGGCCTTGCGGGAGGTTTGGGCCAGGATCCTTCCCGAGAGAGAGGACCCCCTTGTCC2040              TGGGCAGCTGTCACTGGGGTAGCCTGTTTTGGAAGTGTGCGGGCAAGCGTTCGAGCTGCC2100              CCATTGGGGGCGCTATTAGAACACTGCAGCGCGAACGTGAAGATCTTTTTCTCTACTTAT2160              CCCTACTTCCAAAATGTAAATTTGCGCCCCTTGGTGACTGTCCGCCCTTGGTTTGGCCCT2220              GCATGTTGCAGACCTCATCTCCTACCCACCCGTAATTACCCCCCCAACCAGGACAGGTCT2280              GGGCCCGGAACTAGAGCCTTAGGCTAGAGTTAGGGAGGGGGCGGCTACAGGAATTGGTGT2340              TCGGGCCTCGAGCCGTCCCGCGGGCCTGACTCAGTCGCCCTTCTGTTTGCAGATGGACTA2400              CAGCGGCCCCCCGAGCGGCGCCCGGCGGCGGAACTGCTACGAAGGCGCCTACTACAACGA2460              GGCGCCCAGCGGTGGGTATTCCGGGCCTCTCCCTGCTCGCTCCTCCTCCTTCATGGAGCT2520              GTCCTGGCCTCTATCTAGGACGCTCCCACCCCCACTCACACACGCCTATGTCCTGGGAAG2580              TGGTGCAGGAGATGAAATACTAAGCAAGTAGCTCCCTGTCTTTTCGATTGTCCCGGACTC2640              TAACTAAAGTCCTCAGTTTCCAATCTGTCTCAAAGTACTGGGCCCGGGGGTGGGAGGCTT2700              GTCGCGGCCCCACCCCTGCTTACTAACCGAGCCCTCCCCGCGCAGAACCCAGGCCCGGGA2760              AGAGTGCGGCGGTGTCGAGCCTAGACTGCCTGTCCAGCATCGTGGAGCGCATCTCCACCG2820              AGAGCCTGCGGCGCCCGCCCTCCTGCTGGCGGACGTGCCTTCTGAGTCGCCTCCGCGCAG2880              GCAAGAGGCTGCCGCCCCCAGCGAGGGAGAGAGCAGCGGCGACCCCACCCAGTCACCGGA2940              CGCCGCCCCGCAGTGCCCTGCGGGTGCGAACCCCAACCCGATATACCAGGTGCTCTGAGG3000              GGATGGTGGCCGCCCACCCCAACCCCGCCCGAGGGATGGTGCCCCTAGGGTCCCTCGCGC3060              CCAAAAGATTGAACTTAAATGCCCCCCTCCCAACAGCGCTTTAAAAGCGACCTCTCTTGA3120              GGTAGGAGAGGCGGGAGAACTGAAGTTTCCGCCCCCGCCCCACAGGGCAAGGACACAGCG3180              CGGTTTTTTCCACGCAGCACCCTTCTCGGAGACCCATTGCGATGGCCGCTCCGTGTTCCT3240              CGGTGGGCCAGAGCTGAACCTTGAGGGGCTAGGTTCAGCTTTCTCGCGCCCTCCCCATGG3300              GGGTGAGACCCTCGCAGACCTAAGCCCTGCCCCGGGATGCACCGGTTATTTGGGGGGGCG3360              TGAGACCCAGTGCACTCCGGTCCCAAATGTAGCAGGTGTAACCGTAACCCACCCCCAACC3420              CGTTTCCCGGTTCAGGACCACTTTTTGTAATACTTTTGTAATCTATTCCTGTAAATAAGA3480              GTTGCTTTGCCAGAGCAGGAGCCCCTGGGGCTGTATTTATCTCTGAGGCATGGTGTGTGG3540              TGCTACAGGGAATTTGTACGTTTATACCGCAGGCGGGCGAGCCGCGGGCGCTCGCTCAGG3600              TGATCAAAATAAAGGCGCTAATTTATACCGCCGTGGCTCCGGCTTTCCCTGGACATGGGT3660              GTGGGATCCGGAGGAAAATCCGCAAACTGGGCCAGCTGTCCCTCAGCGACGCCTGTAGGC3720              GGCAGGCGGATTGCAAGGAGGAAGCCTGCTGCCTGGGGAAGGAAGGAGGGGTGCAAATTT3780              CTCCAGTACGTGAGGAAGTTCCTCTGACCTTGACTACATTACTACACACGTCCGTGGCTC3840              TTATGGAAGGGTACACAGGTTGATATGAGTATTTTTTAAACCCATGTCTGAGCTCGCCCC3900              CTAGATATTCTGATTTAATGTTTCTGCCCCATATACCCAGGGCCAGGTATTGGTATTTTT3960              TTTCAAAAGCTCCCCAAGTGATTCTGAAGTTCATTCAAGGCTGAGAATCATCCCTCCATA4020              TAAGTGAGTGAACCCAGGTGTGATACAGAGACACGGAGTGTGCCAGGCATCACTTGGGGC4080              TCGTGG4086                                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1757 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCACAGCAGTTGGGGGCATTTATGGGCCTTCCTATAAACTTCTGAGAGGGTAACTTTATC60                CTGCTTCTTTCAGCCAAGTATCCTCCTCCAGCAGCTGGTCACAAAGCTGGTTAATCTCCC120               AGAGTGCTCAGCTTAAAACCCGTGACTCACAGCACAGCCAGTGTGGGGGAGGGGGTGGCT180               GCCTCCAATACGTGGCGCCCAGAGTCAGCTGTTCTGGGGCCTTCTCTGGTTTCTCCAACT240               GAGTCCTGAGGTTTGGGGCCTTGTCTTCCTTCCTGGAGTCCTGCTTCTCACTGACCCCTA300               CATACAAGCCATGAGAGGTCAGGGACCTGAGAGGAGGGCCAGTTCCAGGCCTTGGCTTTG360               GCCAAGCCCTCAGGCTATCCCAGAAATGACCAGAAGGCCTTGGCCTTCCAGAGAAGGGGA420               AGGTTTCAAGTGTAACTCTGGGAGGGGTTGGTCCTGAAATTGGGGTCCCTGCCTCACCTG480               CCCAGACCTGGAAAAATTCCCTTCAGCCATGACCCTCTCATGGCGGATCTTCATTCCCTG540               TCAGCATGTGACATGAAACCTGTGTATGGTGGCTGAAGTGAGCTAGCAAAAAGTAACACA600               AATGACAGGGGACCTCTGACTTGAGATCAGCAGAATAAACACAAGTCGAGTCAGGTAGAA660               AAGGTGGAGTAGTGTTTTGGCCTTGGAGAGACATGGGTTCAAGTCCCAACTCTGCCACCT720               ACTAGCTGAATAGCTTCCCTGAGCCTCTGTTTCCTCCTCTGTAAAACTGGGATAGTAATA780               GCATTACCTTGGCGAGCTAATGTGAGAATCAAAACCTATTTTCCTGCTTAGTAGGTGGGA840               GCTATTAATATTATTGTTGTTATCGTCATCATCATACTGCTCAAAAAGCAGGAGAATCCA900               TTTTCATTTGTCAGGGGACTTATGTTTGTATAGCGGGGAGGGAAGCTAATGGTCTGAAAG960               GATTTCAGTGACACCTCTCACTTGGCAGGAAATCTATTCTGATGAATATGACTCTGTAAA1020              TGATAAGGGAGTATCTGCCAGCCAGTGGCATCGTGCTTGTTATGGTTGAAGACCTAACCC1080              AGGAAACAGCTATAGCAGATACACGACGGAGGCTCCCACTGGTACCTCTACTGAGCAAAG1140              CACAAATCGTGTGCTAACCCTTGCTCCTGTGGTGCCAGTGATTCTCAATACCTTCTACTC1200              CATCTGAAAAGTCCCATACTCATCCAAAGATTCCTGTGTGTAAGGAGGAATGAACCACTT1260              TATAAGTTCCTGTTATGGGCCAGACACTATATTAAACACAAATATTTGACCATATCTAAC1320              CCTTACAACATCCCTTGGAGTGGGTATACTATTATCTACATGTGGTGGACCAATTATATT1380              AATGAATCTAGTTCTTCACTCCTCCTCGTATTCATACCCTTTGCCTTATGATTTTGCAAC1440              TCTTCATATCAGGAGGCATATTGTGTATTTCTCCATGTCTCAGTTCTGAGTTCAGCCATG1500              TAACTTGTTTTAACCCATGAGATATTAACATATATGAATCAGGCAGAGGTTTGGGAAATG1560              TGCTTATGTTTCTGCTTGCACTTTTGCACCACTACCATTACCATGAAAACACGCCTAGGC1620              TAGCCTGCTAGAGGTGAGGCCTGTGGAGCGCAGCTGAGTCGCCCAGTTCCCCAGCCAAGA1680              CCAGCCTGAGCCAGTAAAGTACAGCATGTGAGTGAGCCCAGCAGAGCCTAGGAAAACAGA1740              CCAATCTAAATAGCCAA1757                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2235 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: Not Relevant                                                    (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Quail (Coturnix spp.)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACGTTGTCAAGGAAAATTCTGAGTCTTTTTTAAAAGTGAAAGCCAACACAGTAGCACTGA60                CACTTGTGTGTATTTGTGGTGAGGTCAATGACTGTTATGGATTTTAGACTGTTTTTTTTC120               TGCCTGTGCCATTCTGGCTACCACCTCTGCTCCTTGAAGTGACTCTGCTTTGCTTCTTTC180               TGTAATATATCCCATCTGGACATGGTCCAAGTGGAAAGTGACTCAAAACTAATCAAACCT240               CAGAAGGTCAAAANTGAAAAGAGGTACAGTTCAGGGAAATACATGTTAGAATACGTGTTA300               GAGAAGTTGTGACTGGTGATATGAGCAGCTTGTAGCAAGGTCATGTTTTCCCCTAACACT360               GCTTTGTGAGCACTTTGGAAAGCCTACTTTTGCTCAGGTTTTGTCTGATGTGTCCCAGAA420               CGGGATCATCCATATTTCCTTGAAGGATGCTTATGGTCTAGAATCTGGGATGCAAACAGG480               ACTGAGGGACACATCTTGTGAGGCAGCAGTAAGGCCATGGTACGTGGGCAGAGGGAGGGT540               AGTGAAGTTTGCCATGTGTAGCTTTTGACTTGTAGCTGTNTGCTTTGAAGCAGGAGACAA600               GAAGATTTATTTTCCTTTTTGAAGGAAGATCAGTGCACAGCAAAGATAGGTGAGAAGTTC660               CAAGGAAAACTAAACAGAGAAGAGAAGCAGCACTAACTGGCAGAGTGGGCCAAACCTTTC720               ACTGTTGTATATGGGCATTACTCATACAACTTCAAGAGAGTACATGATTGAACTGAGCAT780               GTACCAGCTGAGGGCCTGGCCCATAATGTTCNTTATAAAGGTCCGATTCCTCCCCAAATA840               GTTTTTCCCTCTCTCTTCAAAGGGGCACCTGTTGTTGGAGGAGCTGGTGATGATACTGGA900               TTAGTGCACATGCGGTCAGCCCACTTGGCCTCGGCCCTTTGGACCCAAAATGAACTCCAG960               CTGCTGTTACCCAGAGCAGGTGCTTCATCCAGCCTGTGCAGCTGTTTGAATGCATGCTGT1020              TGTGGCCAATAGGCGGGGTGAGTCCTCTGAACTACCAGGGGAAGAGCTGGTCAGCAGGAG1080              GGAAGGGAAAGGCACAGAGCTGGGTTTCTTATACCCAGCATTTAGCAAGGAGACAGTGTT1140              CCAGCATAGTATGGTGGAAATGGGAAACAGTGGCTGGTATCCTGCATGCAACATGCCCAC1200              ATGACCCAGTGATGGATGCTTGTTCCCAAAATGAGGCTGAGACCTATAGAATACCAGCAG1260              GACCCTGACAAATGCTGGATCTGTAAGATGCTGAATCTCCCTTGTCAGTTACTGGCCTAG1320              TGTGAGACATTCAGAGGGCTGCTGGCATCTAACAGTTACTCAGTGTTTTCAGCCACTGGT1380              TTAAAGCTTTAAAGAGCTGCCTGGCGAAGGTGAGATAGGCGCAGAGCGCGTGCAGGGTGA1440              ATATCTGTGACGTGCAANAGCTGAAACCAGCAGCAAAGGAAGATGACAAAAGCAGAGGGA1500              AATGGGTTAAGATGCAGCCACGGGAGTGCAAGGGACTGTGCCAGGTCAGTGGAGGGTGAG1560              GAGACNCGGGCGTTCAGAGTTAGGGAAGGCTGGAAGTCAGCAGCCAGAGTTTGAAGAAGG1620              AGTATAGACAGGTAACACCAATGGTAGAGCAGTGGTAACCCAGGGAGGGNNAGAGAGAAG1680              GGAGCAGGGCAGGNNTGAAGGTTTCTTTTTTCTACATTGCATATGGTTTCAGTCAGGTCT1740              CATCAGCCAGGCTTCTCATTCTTCATGCCTTTGCTAATTGCTCAAGCAAGCTCTCAGCGA1800              ACCTCCATATTTCATTTTTCATTACAGTGTGGCGCAAGCCCAGGAGAAAAACATAAATAT1860              TTGAGGCCTCTCTTTGTCAGGAAATGGGATTTCNGCAGGTGCTCATTTGCAAATACTGTG1920              CATGCTTCTGAGGCTTGGNATANGGCATTGCTAAATCCTGATTCAGGATGCAAGAATGTC1980              TCGTGGCCTCTGCCATGTAAACTGTTGTCCGCCCAAGTTTGGAAGTCAGCCCTCAGTGAT2040              GGCACTAGACAAGTATGGGTGAAATGAGCAGCTTGGCTTCAGCACTGAGCAAGACTTGTT2100              AAACACTGTAAGTACAGATGGGCCAATTCACAGTTTGAATAGTATAACAATACATATATA2160              TATAATATTATGGCTTTTTCTGCAGGNNNTCGANNNNANNNNNNNCGATACCGACGACCT2220              CGAGGGGGGCCGGTA2235                                                           __________________________________________________________________________

What is claimed is:
 1. A DNA segment isolated from the region 10-30 kb5' of the coding region of the human myoD gene having an enhanceractivity in non-differentiated cultured skeletal muscle cells and innon-cultured skeletal myogenic cells, wherein said enhancer activitycauses increased expression of a target gene when said DNA segment andsaid target gene are disposed within a DNA strand and said DNA segmentis so positioned in the 5' direction relative to said target gene topermit said increased expression of said target gene.
 2. The isolatedDNA segment according to claim 1, positioned within 100 kilobases (kb)in the 5' direction of said target gene.
 3. The isolated DNA segmentaccording to claim 2, positioned between 10 kb and 30 kb in the 5'direction of said target gene.
 4. The isolated DNA segment according toclaim 1, isolated from a region approximately 18-22 kb in the 5'direction from said human myoD gene.
 5. An anti-sense oligonucleotidehaving a sequence that hybridizes with the DNA segment according toclaim
 1. 6. A vector comprising the DNA segment according to claim
 1. 7.A procaryotic or eucaryotic host cell transformed or transfected withthe vector according to claim
 6. 8. An isolated quail qmf1 DNA segmenthaving an enhancer activity in cultured skeletal muscle cells and innon-cultured, skeletal myogenic cells, wherein said enhancer activitycauses increased expression of a target gene when said DNA segment andsaid target gene are disposed within a DNA strand and said DNA segmentis so positioned in the 5' direction relative to said target gene topermit said increased expression of said target gene.
 9. The isolatedDNA segment according to claim 8, isolated from a region approximately11.5-15 kb in the 5' direction from said qmf1 gene.
 10. A DNA segmentisolated and purified from an approximately 25.5-kb fragment adjacent inthe 5' direction to a human myoD gene, having a nucleotide sequencesubstantially the same as Sequence I.D. No. 2, described herein.
 11. Avector comprising the DNA segment according to claim
 10. 12. The DNAsegment according to claim 10, consisting essentially of a nucleotidesequence substantially the same as bases 1-258 of Sequence I.D. No. 2,described herein.
 13. The DNA segment according to claim 10, consistingessentially of a nucleotide sequence substantially the same as bases1185-1757 of Sequence I.D. No. 2, described herein.
 14. A DNA segmentisolated and purified from an approximately 18-kb fragment adjacent inthe 5' direction to a quail qmf1 gene, having a nucleotide sequencesubstantially the same as Sequence I.D. No. 3, described herein.
 15. Avector comprising the DNA segment according to claim
 14. 16. An isolatedDNA segment having an enhancer activity in cultured cells, wherein saidenhancer activity causes increased expression of a target gene when saidDNA segment and said target gene are disposed within a DNA strand andsaid DNA segment is so positioned in the 5' direction relative to saidtarget gene to permit said increased expression of said target gene,said DNA segment being isolated by a method comprising:a) obtaining atleast one test segment comprising DNA sequences disposed in the 5'direction 10-100 kb of a gene encoding a bHLH myogenic regulatoryprotein, one or more of said at least one test segment being suspectedof having said enhancer activity; b) preparing a set of test constructs,each said test construct comprising one said test segment, a reportergene and a vector adapted for expression in a cultured eucaryotic cell,said test segment and said reporter gene being so located relative toeach other and to regulatory sequences of said vector to permitexpression of said reporter gene, as well as said enhancing activity, ifpresent, of said test segment; c) preparing a control constructcomprising a reporter gene and a vector adapted for expression in acultured eucaryotic cell, said reporter gene being so located relativeto regulatory sequences of said vector to permit expression of saidreporter gene; d) introducing each said test construct or said controlconstruct into cultured eucaryotic cells under conditions permittingexpression of said reporter gene, said expression causing formation of adetectable product in an amount correlatable to said expression; e)establishing a ratio of said amount of detectable product formed in saidcultured eucaryotic cells comprising said test construct to said amountof detectable product formed in said cultured eucaryotic cellscomprising said control construct, the magnitude of said ratio beingindicative of said enhancer activity suspected of being possessed bysaid test segment; and f) identifying each said test segment possessingsaid enhancer activity, thereby isolating said DNA segment having saidenhancer activity.
 17. An isolated DNA segment having an enhanceractivity specifically in myogenic cells of a living animal, wherein saidenhancer activity causes increased expression of a target gene when saidDNA segment and said target gene are disposed within a DNA strand andsaid DNA segment is so positioned in the 5' direction relative to saidtarget gene to permit said increased expression of said target gene,said DNA segment being isolated by a method comprising:a) obtaining atleast one test segment comprising DNA sequences disposed in the 5'direction within 100 kb of a gene encoding a bHLH myogenic regulatoryprotein, one or more of said at least one test segment being suspectedof having said enhancer activity; b) preparing a set of test constructs,each said test construct comprising one said test segment, a reportergene and regulatory sequences necessary for expression of said reportergene in cells of a vertebrate embryo, said test segment and saidreporter gene being so located relative to each other an to saidregulatory sequences to permit expression of said reporter gene, as wellas said enhancing activity, if present, of said test segment; c)introducing each said test construct into cells of said vertebrateembryo under conditions permitting expression of said reporter gene,said expression causing formation of a detectable product in an amountcorrelatable to said expression; d) determining which, if any, cells ofsaid vertebrate embryo form said detectable product, the formation ofsaid detectable product specifically in myogenic cells of saidvertebrate embryo being indicative of said enhancer activity; and e)identifying each said test segment possessing said enhancer activity,thereby isolating said DNA segment having said enhancer activity.